BoneKEy-Osteovision | Commentary
Regulation of tartrate resistant acid phosphatase (Trap) in osteoclasts
DOI:10.1138/2001050
Trap (tartrate resistant acid phosphatase) is a widely used marker of osteoclastic phenotype. Since the study of Minkin () it has served as a cytochemical marker for osteoclasts and has helped a number of researchers to identify osteoclasts and their precursors in tissue sections as well as in cell cultures. More recently serum levels of active Trap5b have been successfully used to measure bone resorption activity in various metabolic bone diseases in humans ().
In spite of the intensive use of Trap as a cytochemical marker for osteoclasts the function of Trap is still poorly understood. There is solid proof that it could function as a protein tyrosine phosphatase but so far no such substrates have been identified. The other function for Trap could be a generation of reactive oxygen radicals via Fenton's reaction in the catalytic center containing two iron atoms ().
TRAP null mutation actually created more questions than answers concerning the cell physiological function of this protein (). However, the skeletal phenotype of Trap-/- mice suggests that Trap is needed for the optimal bone turnover.
The regulation of Trap expression in osteoclasts is also interesting from the point of view of osteoclast differentiation. Understanding the regulation of Trap expression at the transcriptional level should also enlighten the whole process of osteoclast differentiation. Two recent papers, one by Matsumoto et al. () and another by Luchin et al. () could be important achievements to clarify osteoclast differentiation as well as an example how lineage-specific gene regulation can be achieved by the combinatorial action of non-tissue specific transcription factors.
Luchin et al. () first demonstrated that one of the Ets family transcription factors, PU.1 specifically binds to Trap promoter. The binding site for PU.1 (GGAA) is located only 10 base pairs upstream of the binding site for microphthalmia transcription factor (MITF) which is a basic helix-loop helix zipper protein and carries mutations in the human genetic diseases Waardenburg's syndrome 2A and Tietz syndrome. In the mi/mi rat there is a homozygous deletion is in the MITF-locus. Previous studies have further shown PU.1-/- mice do not have either Trap positive mononuclear or multinuclear osteoclasts () and that mi/mi mice have a lot of small and Trap positive osteoclasts ().
After recognising that PU.1 and MITF binding sites are close to each other in the Trap promoter, Luchin et al. continued by showing that the expression of either PU.1 or MITF alone caused only 4-5-fold activation of the Trap reporter gene but that the simultaneous expression of these two transcription factors induced a 20-fold activation. The authors went further on and showed a physical interaction between MITF and PU.1. However, activation was not due to the cooperative binding of transcription factors to the Trap promoter. Final proof for interaction of MITF and PU.1 was demonstrated by producing double heterozygous MITF/mi/PU.1+/- mice. Mice heterozygous for either the mi allele or PU.1 knockout allele do not have skeletal phenotype but approximately 25 % of double PU.1/mi heterozygous mice are clearly osteopetrotic.
Masahito et al. () in their paper found another partner to MITF, namely PU.1-interacting protein, Pip, which synergistically enhanced the promoter activity of Trap gene. An interesting thing is that previous studies have shown that PU.1 recruits binding of Pip to the transcriptional complex. They first demonstrated that, in addition of Trap expression, RANKL increases the expression of Pip in macrophages without having effect on other members of IRF family proteins. When the IRF-E site was deleted from Trap promoter construct no Pip induced enhancing activity was observed. In contrast to Luchin et al. (), Masahito et al. found that PU.1 had no effect on the expression of Trap in HEK 293 cells. This discrepancy is difficult to explain since both groups found similar activation of Trap promoter with MITF alone. Thus it remains to be seen if Pip regulates Trap promoter only via direct binding to IRF-E site or if there is an additional pathway via interaction with PU.1.
The promoter region of Trap contains binding sites for several transcription factors, e.g. AP-1, PU.1, IRF-E and M-box. If we learn how Trap expression is regulated at the transcriptional level we will also know much more about the osteoclast differentiation.
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